Ischemia and reperfusion (I/R) injury of the brain occurs following resuscitation from cardiac arrest and stroke, and results in high morbidity and mortality. There is no clinically effective treatment because of an incomplete understanding of the cellular injury cascades leading to cell death. The long- term goal of my laboratory is to investigate the mechanisms of neuronal death caused by brain I/R to allow for the development of effective treatments. There is a striking correlation between protein synthesis inhibition and the selective death of hippocampal CA1 pyramidal neurons following transient global brain I/R. The mechanism of this irreversible translation arrest and its relationship to cell death is unknown. Stress granules are cytoplasmic particles that sequester inactive translational machinery during cellular stress. We present compelling evidence that stress granule alterations are central to persistent translation arrest in ischemic-vulnerable hippocampal CA1 neurons. Our Specific Aims are: 1. To investigate the mechanism of irreversible translation arrest. We will analyze the functional composition of stress granules utilizing complementary microscopic and biochemical approaches. We will compare stress granules in ischemic resistant CA3 and ischemic vulnerable CA1 from early reperfusion to the point of cell death of vulnerable neurons. 2. To identify the effect of ischemic preconditioning (IPC) on stress granule composition and behavior in reperfused neurons. IPC prevents both cell death and persistent translation arrest in vulnerable CA1 neurons. We will assess the effect of IPC on stress granule behavior and composition, protein synthesis rates, and cell death in CA1 neurons. 3. To show that persistent translation arrest is causally related to neuronal death following brain I/R. Antibiotic protein synthesis inhibitors will be used to predictably alter stress granules in reperfused hippocampal neurons, and we will examine the effect on protein synthesis rates, stress granule composition and behavior and cell death in reperfused hippocampal neurons. By providing an integrated examination of the relationship between persistent translation arrest and I/R-induced cell death, our Specific Aims address a problem that has been a barrier to progress in the field: how irreversible inhibition of protein synthesis in reperfused neurons causes cell death. PUBLIC HEATH RELEVANCE: Every year, millions of people are injured or die from brain damage caused by cardiac arrest or stroke. There are no treatments to prevent this brain damage because physicians and scientists do not understand how the cells die following a period when blood has stopped flowing in the brain and subsequently resumed. The work in this proposal seeks to further our understanding of the way in which neurons in the brain die following a period of low or no blood flow (ischemia) followed by resumption of normal blood flow (reperfusion).